Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome-wide gene expression analysis of the whole blood leukocyte transcriptional response to endotoxin treatment

ABSTRACT: Several inflammatory diseases respond to TNFα inhibitors, implicating TNFα signaling in the pathogenesis of these conditions. Here, we set out to map the systemic genome-wide transcriptome influenced by TNFα release. We performed an intravenous endotoxin challenge in 16 healthy subjects, half of whom were pretreated with the soluble TNF receptor fusion protein, etanercept. Whole-blood leukocyte total RNA was isolated using the PAXgeneTM tube and PAXgeneTM RNA isolation system (PreAnalytiXTM, Qiagen) as described by the manufacturer. Total RNA yield and purity (260nm:280nm) were determined spectrophotometrically. The integrity of the re-suspended total RNA was determined using the RNA Nano Chip Kit on the Bioanalyzer 2100 and the 2100 Expert software (Agilent). To increase the sensitivity of our gene expression assay, the overload of globin mRNA of whole blood samples was reduced by applying the human GLOBINclear kit (Ambion/Appied Biosystems). Synthesis, amplification and purification of anti-sense RNA was performed using 300 ng of enriched mRNA per sample and the Illumina TotalPrep RNA Amplification Kit (Ambion/Applied Biosystems) following the Illumina Sentrix Array Matrix expression protocol. A total of 750 ng biotinylated cRNA was hybridized onto the HumanHT-12v3 expression BeadChips (Illumina). The BeadChips were scanned according to the protocol described in the Illumina Whole Genome Gene Expression for BeadStation Manual v3.2, Revision A using scanning software BeadScan 3.5.31. The GenomeStudio® Data Analysis Software (Illumina) was used for data collection. Missing values were imputed by using the k-nearest-neighbor algorithm (k=15). The raw scan and imputed data were read using the lumi available through Bioconductor. This was carried out using the R statistical package (version 2.10.1; R Foundation for Statistical Computing, Vienna, Austria). Quality control checks of the raw non-normalized data included visualization of the similarity matrix and hierarchical clustering analysis. Samples for one placebo-treated individual did not pass our quality control checks, and thus were removed from subsequent normalization and analysis. Total RNA isolated from a collection of human tissue-derived cell lines was used as an internal control; this sample also was omitted from subsequent data normalization. Variance stabilizing normalization (Biconductor library vsn) was applied to all other samples. All subsequent analyses were performed on vsn-transformed intensity values. This study investigated the genome-wide transcriptional response to endotoxin-induced TNF-alpha-mediated signaling in whole-blood leukocytes. Total RNA was isolated from PAXgene-treated whole blood of 16 healthy volunteers before and 4 hours after infusion of 1ng/kg E. coli lipopolysaccharide. Eight of these subjects were treated with the TNF-alpha inhibitor, etanercept.

ORGANISM(S): Homo sapiens

SUBMITTER: Brendon Scicluna

PROVIDER: E-GEOD-36177 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Role of tumor necrosis factor-α in the human systemic endotoxin-induced transcriptome.

Scicluna Brendon P BP van 't Veer Cornelis C Nieuwdorp Max M Felsmann Karen K Wlotzka Britta B Stroes Erik S G ES van der Poll Tom T

PloS one 20131113 11

TNFα has been implicated in the pathogenesis of various inflammatory diseases. Different strategies to inhibit TNFα in patients with sepsis and chronic inflammatory conditions have shown contrasting outcomes. Although TNFα inhibitors are widely used in clinical practice, the impact of TNFα antagonism on white blood cell gene expression profiles during acute inflammation in humans in vivo has not been assessed. We here leveraged the established model of human endotoxemia to examine the effect of ...[more]

PMID: 24236088

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Project description:Several inflammatory diseases respond to TNFα inhibitors, implicating TNFα signaling in the pathogenesis of these conditions. Here, we set out to map the systemic genome-wide transcriptome influenced by TNFα release. We performed an intravenous endotoxin challenge in 16 healthy subjects, half of whom were pretreated with the soluble TNF receptor fusion protein, etanercept. Whole-blood leukocyte total RNA was isolated using the PAXgeneTM tube and PAXgeneTM RNA isolation system (PreAnalytiXTM, Qiagen) as described by the manufacturer. Total RNA yield and purity (260nm:280nm) were determined spectrophotometrically. The integrity of the re-suspended total RNA was determined using the RNA Nano Chip Kit on the Bioanalyzer 2100 and the 2100 Expert software (Agilent). To increase the sensitivity of our gene expression assay, the overload of globin mRNA of whole blood samples was reduced by applying the human GLOBINclear kit (Ambion/Appied Biosystems). Synthesis, amplification and purification of anti-sense RNA was performed using 300 ng of enriched mRNA per sample and the Illumina TotalPrep RNA Amplification Kit (Ambion/Applied Biosystems) following the Illumina Sentrix Array Matrix expression protocol. A total of 750 ng biotinylated cRNA was hybridized onto the HumanHT-12v3 expression BeadChips (Illumina). The BeadChips were scanned according to the protocol described in the Illumina Whole Genome Gene Expression for BeadStation Manual v3.2, Revision A using scanning software BeadScan 3.5.31. The GenomeStudio® Data Analysis Software (Illumina) was used for data collection. Missing values were imputed by using the k-nearest-neighbor algorithm (k=15). The raw scan and imputed data were read using the lumi available through Bioconductor. This was carried out using the R statistical package (version 2.10.1; R Foundation for Statistical Computing, Vienna, Austria). Quality control checks of the raw non-normalized data included visualization of the similarity matrix and hierarchical clustering analysis. Samples for one placebo-treated individual did not pass our quality control checks, and thus were removed from subsequent normalization and analysis. Total RNA isolated from a collection of human tissue-derived cell lines was used as an internal control; this sample also was omitted from subsequent data normalization. Variance stabilizing normalization (Biconductor library vsn) was applied to all other samples. All subsequent analyses were performed on vsn-transformed intensity values.

Project description:The main aim of the study was to compare genome-wide gene expression profiles obtained from the two widely used commercially available whole blood RNA collection systems - PAXgeneTM and TempusTM tubes. Comparisons of present call rates, variances, correlations and influence of globin reduction across the two collection systems was performed using in vivo glucocorticoid stimulation in 24 peripheral blood samples from three individuals. Three healthy male volunteers aged 30-35 years with body mass index of 20-25 were enrolled in the study.1.5 mg dexamethasone stimulated peripheral blood samples were drawn 3 hours later. For each sample, duplicate measures of 2.5 ml and 3ml blood was collected in PAXgeneTM tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland) and TempusTM tubes (Applied Biosystems, Foster City, CA, USA) respectively. The PAXgeneTM and TempusTM tubes were stored for 2.5 hours at room temperature and then transferred to -20°C. Due to the lower RNA yield from the PAXgeneTM tubes, RNA from two PAXgeneTM tubes was pooled together for the experiment . Total RNA was isolated from whole blood stored in PAXgeneTM and TempusTM tubes according to the respective manufacturer's instructions. The PAXgeneTM samples were processed using the PAXgeneTM Blood RNA Kit based on the Quiagen method for column purification of nucleic acids (PreAnalytiX GmbH, Hombrechtikon, Switzerland, catalogue number 762174). The TempusTM samples were isolated using the TempusTM Spin RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA, catalogue number 4380204). RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The concentration and purity of total RNA was independently assessed by 260/280 UV absorption ratios, respectively (Nanophotometer, Implen, Munich, Germany). RNA samples were divided into non-globin reduced and globin reduced groups. The GLOBINclearTM-Human Kit (Ambion, Inc., Texas, USA, catalogue number #AM1980) was used to remove globin mRNA. Sample amplification and labeling for both globin-reduced and non-reduced samples was performed using the IlluminaR TotalPrep RNA Amplification Kit (Ambion, Inc., Texas, USA, catalogue number AMIL1791).

Project description:Background: Although TNF inhibitors are used to treat chronic inflammatory diseases, there is little information about how long-term inhibition of TNF affects the homeostatic functions that TNF maintains in the intact CNS. TNF is known to modulate neurogenesis by decreasing cell proliferation, increasing apoptosis of precursor cells, and impairing neuronal differentiation. TNF can also influence the formation of the hippocampus, with long-lasting effects on cognition. Materials and methods: To clarify whether developmental TNF deficiency causes alterations in the naïve CNS, we estimated the number of proliferating cells, microglia, and neurons in the brains of E13.5, P7, and adult TNF +/+ and TNF-/- mice and measured changes in gene and protein expression and monoamine levels in adult TNF+/+ and TNF-/- mice. To evaluate long-term effects of TNF inhibitors, we treated healthy adult C57BL/6 mice with either saline, selective soluble TNF inhibitor XPro1595, or nonselective TNF inhibitor etanercept. We estimated changes in cell number and protein expression after two months of treatment. We assessed the effects of TNF deficiency on cognition by testing adult TNF+/+ and TNF-/- mice and anti-TNF treated mice with behavioral tasks. Results: TNF deficiency initially decreased the number of proliferating cells and microglia, but subsequently increased the number of neurons. At the same time, TNF deficiency decreased the expression of WNT signaling-related proteins, specifically Collagen Triple Helix Repeat Containing 1 (CTHRC1) and Frizzled receptor 6 (FZD6). In contrast to XPro1595, longterm inhibition of TNF with etanercept in adult C57BL/6 mice decreased the number of proliferating cells in the granule cell layer of the dentate gyrus. Etanercept, but not XPro1595, also impaired spatial learning and memory in the Barnes maze memory test. Conclusion: TNF deficiency impacts the organization of neurogenic zones and alters the cell composition in brain. Long-term inhibition of TNF with the nonselective TNF inhibitor etanercept, but not the soluble TNF inhibitor XPro1595, decreases neurogenesis in the adult mouse hippocampus and impairs learning and memory after two months of treatment.

Project description:The success of TNF inhibitors for treatment of psoriasis and other inflammatory diseases was previously attributed to blockade of innate immunity. In a clinical trial using etanercept TNF blocking agent to treat psoriasis vulgaris, we used affymetrix gene arrays to analyze broad gene profiles in lesional skin at multiple timepoints during drug treatment (baseline, and weeks 1, 2, 4 and 12) compared to non-lesional skin. This analysis created a temporal model of TNF-dependent gene regulation that informs molecular mechanisms of TNF-mediated inflammation. We identified four gene clusters that were differentially down-modulated during etanercept treatment: the cluster down-regulated most rapidly contained mostly dendritic cell activation genes. Culturing human keratinocytes with TNF, IFNg and IL-17 generated a list of keratinocyte genes regulated by each cytokine. The IL-17 pathway genes were strongly down-modulated early, whereas IFNg pathway genes were not down-modulated until final disease resolution at week 12. Finally, we show that TNF blockade rapidly inhibits IL-12/IL-23 p40 subunit expression, and that p40 neutralization inhibits psoriatic dermal emigre-mediated Th17 polarization. We hypothesize that etanercept inhibits myeloid dendritic cell production of IL-23, a Th17 survival cytokine, resulting in rapid downregulation of IL-17 pathway genes. This data links effects of TNF blockade on the innate immune system with the adaptive immune system. Experiment Overall Design: In this study 15 patients with moderate-to-severe psoriasis were given 50mg of etanercept (Amgen) biweekly for 12 weeks. And analyzed using gene array on mRNA extracted from tissue collected at each biopsy time point (non-lesional Time: 0; lesional Time: 0, weeks 1, 2, 4, and 12). Patients were stratified as 'responders' or 'non-responders' based on whether or not they achieved histologic disease resolution by week 12 of etanercept treatment (decreased epidermal thickening, normalization of proliferation marker Ki67, and loss of differentiation marker K16).

Dataset Information

Genome-wide gene expression analysis of the whole blood leukocyte transcriptional response to endotoxin treatment

Publications

Role of tumor necrosis factor-α in the human systemic endotoxin-induced transcriptome.

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